1. Field of the Invention
The present invention relates to fluorogenic substrates and compositions containing the same useful for detecting the activity of enzymes within intact cells. More specifically, the present invention relates to lysosomotropic derivatives of 2,3-dicyano-hydroquinone (DCH), compositions containing the same and methods employing the same for detecting lysosomal enzymatic activity within intact cells.
2. Description of Related Art
Fluorogenic substrates that can be used to detect the activity of enzymes within intact cells are greatly desired for biochemical investigations of human metabolic storage disorders, and identification of gene transfer recombinants. These genetic diseases are caused by the lack of specific enzymes that normally function within lysosomes, which are intracellular membrane-bound organelles containing a variety of hydrolytic enzymes. While in vitro measurement using tissue or cellular extracts has become routine, the detection of enzymatic activity within living cells has proven to be more problematic. Two notable difficulties are the pH dependence of the fluorescence of some probes at physiological pH values (J. Slavik, Intracellular pH and its measurement, CRC Press, A. Ketyk and J. Slavik eds., Florida, (1989), pp. 87-126) and the undesirable tendency of the fluorescent product to diffuse out of the cells (Musgrove et al, Cytometry, vol. 7, (1986), pp. 347-355). Nevertheless, success has been achieved in some systems for example by cooling of the cells in order to slow efflux of the product, fixing of the fluorescent product as an insoluble precipitate or performing the incubations in microdroplets containing a single cell. A final, and more general, solution is the chemical modification of the fluorophore to obtain the desired properties (Yegoror et al, Anal. Lett., 21, (1988), pp. 193-209; Sherman et al, Anal. Chem., 40, (1968), pp. 803-805). A recent example is the synthesis of the 4-nonyl analog of 4-methyl-umbelliferone, and the study of its .beta.-glucoside in normal and Gaucher fibroblasts (Beiberich et al, Biol. Chem. Hoppe-Seyler, vol. 370, (1989), pp. 809-817).
Other conventional fluorogenic substrates which have been used previously for lysosomal enzymatic activity detection include glycosides and esters of fluorescent phenols such as fluorescein (Jongkind et al, Cytometry, vol. 7, (1986), pp. 463-466) and resorufin (Wittrup et al, Cytometry, vol. 9, (1988), pp. 394-404. However, these conventional substrates are generally not optimal for in vivo measurements of enzyme activity and only a few fluorogenic substrates have been used within intact cells. Therefore, substrates that generate a fluorescent signal at intracellular pH conditions upon enzymatic hydrolysis are theoretically ideal and desirable since they exhibit high sensitivity and allow the living cells to be studied by accurate methods such as fluorescence microscopy. It is also desirable that the substrate exhibit lysosomotropic properties so that it is taken up selectively into lysosomes. Various lysosomotropic agents are known (de Duve et al, Biochemical Pharmacology, vol. 23, (1974), pp. 2495-2531; Firestone et al, J. Med. Chem., vol. 22, (1979), pp. 1130-1133). However, the purposeful combination of a lysosomotropic substructure within a fluorogenic enzyme substrate has not been accomplished.